Abstract
Background: Cell differentiation during development is orchestrated by precisely coordinated gene expression programs. While mechanisms such as the maintenance of pluripotent states are well-understood, others like lineage choice and cell-fate decisions remain poorly comprehended. Given that gene expression is influenced not only by transcriptional control but also by post-transcriptional events, we employed monolayer differentiation protocols to delineate early transcriptional and post-transcriptional events in human embryonic stem cell specification. This involved obtaining representative populations of the three germ layers, followed by sequencing of polysome-bound and total RNAs.
Results: We observed a consistent similar distribution of gene upregulation and downregulation when comparing the transcriptome and translatome during the differentiation of all three germ layers. Notably, certain differentially expressed genes were exclusively detected in the polysome fractions, suggesting active post-transcriptional regulation. Upregulated genes in the translatome more accurately reflected the differentiation process. Additionally, genes such as DLX3, DHFR2, and UNC13D were identified as differentially expressed solely in the polysome fraction, indicating their post-transcriptional regulation during ectoderm commitment. Recruitment of these genes to polysomes was also confirmed.
Conclusions: Substantial post-transcriptional modulation was found during germ layer commitment, emphasizing the translatome reliability in capturing nuanced gene expression regulation. These findings highlight the post-transcriptional regulation's critical role in early embryonic development, offering new insights into the molecular mechanisms of cell differentiation.